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Experimental Therapeutics |
Department of Pharmacy, Center of Drug Research, University of Munich, D-81377 Munich, Germany [V. M. D., A. M. V.], and Institute of Pharmacy, University of Innsbruck, A-6020 Innsbruck, Austria [H. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of m. Most importantly, overexpression of the mitochondria protecting proteins Bcl-xL or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, although the data presented here suggest that helenalin induces a mitochondria-dependent pathway. Thus, helenalin is a promising experimental cytotoxic agent that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Resistance to death receptor-induced apoptosis can be caused by mechanisms such as down-regulation of the receptor and deficient up-regulation of CD95-L,3 as well as mutations within the gene encoding the receptor. Moreover, defects in the apoptotic signaling pathway downstream of the CD95 receptor, e.g., by elevated expression of proteins like FLIP, a homologue of caspase-8 that lacks proteolytic activity, can lead to decreased sensitivity toward apoptotic stimuli (3 , 4) .
Members of the antiapoptotic Bcl-2 family proteins confer protection against most apoptotic stimuli that act via mitochondria. Bcl-2 and Bcl-xL were shown to prevent mitochondrial cytochrome c release and subsequent caspase activation and cell death (3 , 5 , 6) . In a large percentage of human neoplasias, antiapoptotic Bcl-2 proteins were found to be overexpressed or proapoptotic Bcl-2 homologues like Bax that appear to be reduced or functionally inactive (3 , 7) . These alterations in expression or functionality of Bcl-2 family members can render tumor cells more resistant to a wide variety of cell death stimuli, including essentially all classical chemotherapeutic drugs (3) .
An important goal in chemotherapy is, therefore, to find new cytotoxic agents that are able to increase or restore the ability of tumor cells to undergo apoptosis.
In this respect, sesquiterpene lactones are promising compounds of natural origin. They represent a structural class of phytochemicals typically found in the compositae family (Asteraceae). Since decades ago, these plant constituents are known to have cytotoxic capacity. The cytotoxicity of sesquiterpene lactones is suggested to be attributable to their ability to react with sulfhydryl groups, e.g., of cysteine residues in a Michael-type addition (8 , 9) . Thus, the primary cellular target of sesquiterpene lactones differs from those of classical chemotherapeutic drugs like doxorubicin, cisplatin (interaction with DNA), etoposide (inhibition of topoisomerase II), methotrexate (antagonization of folic acid), or vincristine (inhibition of mitosis).
Although the cytotoxicity of sesquiterpene lactones is well documented (8, 9, 10) , it is not known whether sesquiterpene lactones are able to induce apoptosis in tumor cells.
The aims of the present study were, therefore, first, to examine whether helenalin, one of the best investigated sesquiterpene lactones with regard to cytotoxicity (8, 9, 10, 11) , is able to induce apoptosis in leukemia T cells, and, second, to characterize the apoptotic pathways that are involved.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
Human leukemia Jurkat T cells (J16), the CD95-resistant JurkatR (13)
as well as Jurkats transfected with vector control, Bcl-xL or Bcl-2 (Ref. 14
; all kindly provided by Drs. P. H. Krammer and H. Walczak, Heidelberg, Germany) were cultured (37°C and 5% CO2) in RPMI 1640 containing 2 mM
L-glutamine (PAN Biotech, Aidenbach, Germany) supplemented with 10% FCS (PAA Laboratories, Cölbe, Germany). Medium of transfected cells was supplemented with 1 mg/ml G418 (Life Technologies, Inc., Eggenstein, Germany) every fifth passage. Human PBMCs were isolated from heparin-anticoagulated blood of healthy volunteers by centrifugation with Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). PBMCs were stimulated with PHA (1 µg/ml) for 48 h before experimental treatment.
Cell Viability.
Impaired cell viability was measured using the MTT assay, based on the ability of viable cells to reduce yellow MTT to blue formazan, as described previously (15)
. Briefly, cells were exposed for 24 h to helenalin, then incubated with MTT (0.5 mg/ml, 1 h) and subsequently solubilized in DMSO (250 µl) for at least 2 h in the dark. The extend of reduction of MTT was quantified by absorbance measurement (A550 nm).
Detection of Apoptosis.
Apoptosis was judged by (a) cell morphology as described previously (16)
; (b) translocation of phosphatidyl serin to the cell surface using an Annexin V-FITC apoptosis detection kit (Calbiochem, Bad Soden, Germany); (c) the visualization of apoptotic nuclei after staining with HOECHST 33342 (Sigma Chemical Co., Deisenhofen, Germany) by fluorescence microscopy. Quantification of apoptosis was performed according to Nicoletti et al. (17)
. Briefly, cells were incubated for 24 h in a hypotonic buffer (1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml PI) and analyzed by flow cytometry on a FACSCalibur (Becton Dickinson, Heidelberg, Germany). Nuclei to the left of the "G1-peak" containing hypodiploid DNA were considered apoptotic.
Determination of CD95 and CD95-L.
Cells were incubated (4°C, 30 min) with a mouse anti-CD95 antibody (ZB4; Medical & Biological Laboratories Co., Nagoya, Japan), washed two times, and incubated (4°C, 30 min) with a FITC-conjugated rat antimouse IgG1 (PharMingen, Heidelberg, Germany). Cells were washed again and analyzed by FACS. CD95-L expression was measured as described above using a mouse antihuman CD95-L antibody (NOK-1; PharMingen, Heidelberg, Germany), with the exception that cells were fixed in 1 ml of paraformaldehyde (4%; 15 min, 4°C), washed with PBS, permeabilized with ice-cold ethanol (100%, 60 min, 4°C), and washed again prior to incubation with the antihuman CD95-L antibody.
Analysis of Caspase-3-like Activity.
Cells were collected by centrifugation, washed with ice-cold PBS, and lysed in 5 mM MgCl2, 1 mM EGTA, 0.1% Triton X-100, and 25 mM HEPES (pH 7.5) containing the protease inhibitor complete (Roche, Mannheim, Germany). Cytosol was prepared by centrifugation at 14,000 x g (15 min, 4°C). The fluorometric DEVD-afc cleavage assay was carried out according to Thornberry (18)
. In brief, cytosolic extracts (10 µl, approx. 1 mg/ml protein) were diluted 1:10 with substrate buffer [50 µM DEVD-afc in 50 mM HEPES (pH 7.4), 1% sucrose, 0.1% 3-[(3-cuolamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and 10 mM DTT). Generation of free afc at 37°C was determined by fluorescence measurement (SLT Fluostar; SLT Labinstruments, Germany) set at an excitation wavelength of 390 nm and an emission wavelength of 505 nm. Protein concentrations of the corresponding samples were estimated with the Pierce-assay (Pierce, IL), and the activity was calculated using serial diluted standards (0–5 µM afc).
Analysis of 
m.
Cells were stained with the fluorochrome JC-1 (1.25 µg/ml; Molecular Probes, Eugene, OR) according to Bernardi et al. (19)
and Cossarizza et al. (20)
. The membrane potential was measured by FACS. JC-1 aggregates were detected at 585 nm (FL-2), and JC-1 monomers at 530 nm (FL-1).
Measurement of Cytochrome c Release.
Release of cytochrome c from mitochondria was analyzed according to Leist et al. (21)
. Briefly, cell pellets were resuspended in permeabilization buffer [210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 5 mM succinate, 0.2 mM EGTA, 0.15% BSA, and 80 µg/ml digitonin (pH 7.2)] at 4°C and gently shaken at 4°C for 10 min. Permeabilized cells were centrifuged (300 x g); the supernatant was removed, and the cells were centrifuged again (10 min, 13,000 x g). The obtained cytosol was separated on a 15% SDS-PAGE and probed for cytochrome c as described below. The remaining pellet of permeabilized cells was lysed in 0.1% Triton/PBS (15 min, 4°C), centrifuged (12,000 x g, 4°C, 10 min), and the supernatant containing mitochondrial cytochrome c analyzed by SDS-PAGE.
Western Blot Analysis.
Cells were collected by centrifugation, washed with ice-cold PBS, and lysed in 1% Triton X-100, 0.15 M NaCl, and 10 mM Tris-HCl (pH 7.4) with the protease inhibitor complete (Roche, Mannheim, Germany) for 30 min. Lysates were homogenized through a 22-gauge needle and centrifuged at 10,000 x g for 10 min at 4°C. Equal amounts of protein were separated by SDS-PAGE (10% for caspase-8, 12% for Bcl-2 proteins, 15% for caspase-3 and cytochrome c), transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Eschborn, Germany). Equal protein loading was controlled by Coomassie Blue staining of gels. Membranes were blocked with 2% BSA in PBS containing 0.05% Tween 20 (1 h) and incubated with specific antibodies against caspase-8 (mouse monoclonal antibody C-15, 1:5 dilution of hybridoma supernatant; kindly provided by Dr. P. H. Krammer, Heidelberg, Germany), caspase-3 (mouse monoclonal antibody, clone 19; Transduction Laboratories, Heidelberg, Germany), cytochrome c (mouse monoclonal antibody 7H8.2C12; PharMingen, Heidelberg, Germany), Bcl-xL or Bcl-2 (rabbit polyclonal antibodies, clones S18 and N-19, respectively; Santa Cruz Biotechnology, Heidelberg, Germany) overnight at 4°C. Specific proteins were visualized by secondary antibodies conjugated to horseradish peroxidase and the Renaissance Plus reagent (NEN Life Science, Zaventem, Belgium). Pictures were taken on a Kodak Digital Science Image station 440CF (NEN, Life Science, Zaventem, Belgium).
Statistical Analysis.
All of the experiments were performed at least three times. Results are expressed as mean ± SE. Statistical comparisons were made by ANOVA followed by a Dunnett multiple comparisons test or by an unpaired two-tailed Student’s t test. Ps < 0.05 were considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Furthermore, analysis of PI-stained nuclei by flow cytometry showed that helenalin-treated cells displayed a population of nuclei with hypodiploid DNA (Fig. 1B)
. Dose-response studies revealed that helenalin (10–50 µM, 24 h) dose-dependently increased the percentage of apoptotic cells up to 80% (Fig. 2A)
. Helenalin (10 µM)-triggered apoptosis also occurred time-dependently (4–24 h). First significant apoptosis could be detected 16 h after cell stimulation (Fig. 2B)
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Helenalin-induced Apoptosis Is Dependent on Caspase Activation.
To investigate whether helenalin-triggered apoptosis requires the activation of caspases, cells were pretreated with the broad-spectrum caspase inhibitor zVAD-fmk. The inhibitor completely abrogated helenalin-induced apoptosis (Fig. 3A)
which indicated that caspases are essential components in this apoptotic pathway. In addition, helenalin was found to increase caspase-3-like activity time-dependently reaching the level of significance 16 h after cell exposure to helenalin (Fig. 3B)
. To further characterize the involved caspases, the activation of two key components of the caspase cascade, the downstream effector caspase-3 and the initiator caspase-8 were examined. Fig. 3C
shows the time-dependent processing of the inactive Mr 32,000 caspase-3 precursor to the active p17 subunit by Western blot analysis. Cleavage products were detectable as early as 8 h after cell stimulation but increased up to 16 h. Caspase-8, expressed in two functionally active isoforms, caspase-8/a and caspase-8/b, is processed on activation to p43 and p41 intermediates and to the p18 active subunit (22)
. Helenalin also led to activation of caspase-8. Cleavage products of procaspase-8 were detectable 16 h after helenalin treatment, which indicates a delayed activation compared with caspase-3 (Fig. 3D)
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, left). Examining CD95-L expression, no up-regulation could be detected over a period of 24 h. In contrast, helenalin treatment decreased CD95-L expression (Fig. 4A
, right).
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, indicating that apoptosis occurs independently of the CD95 receptor. To further corroborate this outcome, we used an antagonistic anti-CD95 antibody (ZB4). Jurkat cells preincubated for 1 h with the ZB4 antibody did not undergo apoptosis on exposure to soluble CD95-L, whereas on helenalin treatment, antibody-blocked Jurkat cells died similarly to the Jurkat cells that were not preincubated with ZB4 (Fig. 4C)
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Helenalin Induces MMP.
Many apoptotic signals transduce their death-inducing message via mitochondria. A key event in mitochondria-controlled apoptotic pathways is the outer and/or inner MMP, involving the release of proteins, such as cytochrome c, from the intermembrane space and the dissipation of the electrochemical gradient (m) created by the proteins of the respiratory chain located on the inner mitochondrial membrane (23)
. Fig. 5A
shows that, indeed, helenalin caused a time-dependent release of cytochrome c into the cytosol, indicating the occurrence of outer MMP. Considerable amounts of cytochrome c in the cytosol were detectable as early as 8 h after cell treatment. Preincubation with the caspase inhibitor zVAD-fmk (1 h, 50 µM) was unable to prevent cytochrome c release, which indicated that cytochrome c release preceded caspase activation. Helenalin also led to a dissipation of m as detected by flow cytometry using the fluorochrome JC-1 (19
, 20) . Substantial loss of m was evident 16 h after the addition of helenalin as seen by a decrease in FL-2 intensity and an increase in FL-1 intensity (Fig. 5B)
. Pretreatment with zVAD-fmk (1 h, 50 µM) reduced m (Fig. 5C)
, suggesting that caspases are involved in helenalin-induced inner MMP. Thus, helenalin-mediated apoptosis involves inner and outer MMP.
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were exposed to helenalin. Surprisingly, Bcl-xL as well as Bcl-2 failed to abrogate helenalin-mediated apoptotic cell death, although apoptosis was slightly reduced when helenalin was applied at lower concentrations (10–20 µM). Fig. 6B
(top graph) shows the dose-response studies of helenalin performed with Jurkat cells stably transfected with vector alone (Jurkat/neo), Bcl-xL (Jurkat/bcl-xL), or Bcl-2 (Jurkat/bcl-2). To verify that the used transfected Jurkat clones were working properly, we applied staurosporine, known to induce a mitochondrial caspase cascade that is inhibited by Bcl-2 and Bcl-xL (24
, 25) . Fig. 6B
(bottom graph) demonstrates that staurosporine (50–400 nM) dose-dependently triggers apoptosis in Jurkat/neo cells whereas Jurkat/Bcl-xL and Jurkat/Bcl-2 cells are protected against staurosporine. Thus, helenalin seems to be able to trigger apoptosis either by a mechanism that bypasses mitochondrial events or by inactivating effectively mitochondrial protection by Bcl-xL and Bcl-2.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proteolytic cleavage of the initiator caspase-8, which was demonstrated in response to helenalin, typically occurs after triggering cell surface death receptors like the CD95 receptor (5 , 26) . Moreover, in various cell types, chemotherapeutic drugs were demonstrated to activate the CD95 receptor pathway by increasing the CD95 receptor expression or induce the CD95-L (2 , 4) . Therefore, we first investigated whether this mechanism applies also to helenalin. Flow cytometric experiments, however, revealed that helenalin does neither increase CD95 receptor expression nor induce CD95-L. In addition, CD95-resistant Jurkat T cells that lack the CD95 receptor were susceptible to helenalin-induced apoptosis, as are parental cells, indicating that CD95 is not required for helenalin-mediated apoptosis. This result was further corroborated using the antagonistic anti-CD95 antibody ZB4. Preincubation with ZB4 completely blocked CD95-L-induced apoptosis, whereas helenalin-mediated cell death was unaffected. Thus, helenalin is able to trigger apoptosis independent of the CD95 receptor.
Besides the caspase cascades initiated by death receptors, another cascade that is essentially controlled by mitochondria exists (2 , 5 , 7 , 23) . A central event in mitochondria-controlled cell death seems to be the occurrence of outer and/or inner MMP (23) . Outer MMP leads to the release of mitochondrial intermembrane proteins like cytochrome c into the cytosol. Released cytochrome c then triggers the assembly of the so-called apoptosome by interacting with the apoptotic protease-activating factor (Apaf1) and subsequent recruitment of procaspase-9. Processing of procaspase-9 initiates a caspase cascade downstream of mitochondria (5) .
Helenalin, indeed, initiated redistribution of cytochrome c into the cytosol as early as 8 h after cell treatment. Thus, cytochrome c release seemed to be an early event in the helenalin-mediated apoptosis that preceded caspase-3-like activity. Interestingly, the broad-spectrum caspase inhibitor zVAD-fmk was unable to prevent cytochrome c release, which was evidence that caspase-activation occurred solely downstream of mitochondria.
Inner MMP leading to loss of m mostly accompanies outer MMP (23)
. In fact, helenalin clearly induced dissipation of m. Interestingly, loss of m appeared to be delayed compared with cytochrome c release but correlated with the onset of caspase-3-like activity, which suggests a possible link between caspases and mitochondrial perturbation. Indeed, zVAD-fmk was able to considerably reduce dissipation of m. This outcome fits into recently published data proposing a feedback amplification loop between caspase activation and mitochondrial dysfunction (25
, 27, 28, 29, 30, 31)
. This link is mediated either by cytosolic factors that serve as caspase substrates and lead, once cleaved, to cytochrome c release (25
, 28
, 29)
or directly by caspases that affect mitochondrial membrane proteins like the antiapoptotic Bcl-2. Bcl-2 cleavage products then cause cytochrome c release and caspase activation (27
, 30
, 31)
.
Considering our data which strongly implicate a helenalin-initiated caspase cascade downstream of mitochondria, the most interesting finding was that the mitochondria-protecting proteins Bcl-xL and Bcl-2 were unable to prevent helenalin-induced apoptotic cell death. This outcome suggests that helenalin triggers a (second) pathway that bypasses mitochondria and/or that helenalin is able to inactivate the mitochondrial protection conferred by Bcl-2 proteins. Bcl-2 family members are localized at the membranes of mitochondria, in which it is suggested that they control membrane permeability either by collaborating with proteins of the PTP or by forming autonomous pores (7 , 32) . Inactivation of Bcl-2 proteins, e.g., by mutation, phosphorylation, or cleavage can abrogate their mitochondria protective effects (3 , 6 , 7 , 31) . Interestingly, an additional mechanism by which the effect of antiapoptotic Bcl-2 members can be overcome seems to be the thiol cross-linking of the ANT, a protein of the PTP localized on the inner mitochondrial membrane (7) . Sesquiterpene lactones are known to react covalently with thiol groups (8 , 9) . It is, therefore, tempting to speculate on a direct effect of helenalin on the PTP by binding to the ANT. Additional studies will address whether helenalin may also signal independently of mitochondrial events like cytochrome c release or rather whether it targets mitochondrial proteins to overcome Bcl-2 and Bcl-xL-mediated protection.
In view of the apoptosis-inducing potential of helenalin even in Bcl-xL- and Bcl-2-overexpressing cells, it is a remarkable finding that activated PBMCs are resistant toward helenalin. Activated T cells, a major population in PHA-stimulated PBMCs, were shown to possess increased apoptosis resistance by transiently increasing the expression of Bcl-xL (33) . In fact, PHA-activated PBMCs used in the present study were found to express considerable levels of Bcl-xL.4 Bcl-xL overexpression in Jurkat cell, however, failed to confer resistance toward helenalin-induced apoptosis, making it unlikely that Bcl-xL accounts for the remarkable resistance of PBMCs toward helenalin. Other studies suggest that, besides the increase in Bcl-xL, a lack of recruitment of caspase-8 to the death-inducing signaling complex (DISC) might be responsible for the resistance of activated peripheral T cells toward CD95 receptor-mediated apoptosis (34) . Our data, however, clearly demonstrated that helenalin (10 µM) acts independently of the CD95 receptor, although activation of the CD95 receptor at higher concentrations of helenalin cannot be excluded. Very recent data give evidence that activation of the MAPK is involved in the resistance of activated T cells toward CD95-mediated apoptosis (35) . Although the mechanism by which MAPK activation confers resistance to CD95-mediated apoptosis is not exactly known, there are data that point to a direct inhibition of CD95 as well as to a general inhibition of mitochondrial activation, e.g., protection of mitochondria may occur by phosphorylation of the proapoptotic Bcl-2 family member Bad. Phosphorylated Bad dissociates from Bcl-xL, which then promotes cell survival (35, 36, 37) . Thus, MAPK activation may also have an impact on T-cell resistance toward xenobiotic apoptotic stimuli like helenalin. Besides all of these molecular events possibly accounting for the resistance of activated PBMCs toward helenalin, one simple reason might be that these cells are unable to take up this molecule. Additional studies will address this question.
In summary, we found that the sesquiterpene lactone helenalin, which differs in its primary cellular targeting from classical chemotherapeutic drugs, possesses extremely interesting properties with regard to its apoptosis-inducing mechanism. Most chemotherapeutic drugs have been shown to induce apoptosis involving the CD95 receptor system or to affect directly or indirectly mitochondria. However, only a few drugs acting via mitochondria have been shown to overcome the protective effect of Bcl-2 or Bcl-xL. Thiol cross-linkers like diamide, which act on the ANT, or ligands of the mitochondrial benzodiazepin receptor like PK11195 are such compounds (7) . Recently also Tetrocarcin A was shown to suppress the antiapoptotic activity of Bcl-2 by a yet unknown mechanism (38) . Helenalin acting independently of the CD95 receptor, as well as in cells overexpressing Bcl-xL or Bcl-2, thus, may serve as lead compound to develop new chemotherapeutic drugs to overcome chemoresistance attributable to defects in CD95 signaling as well as overexpression of antiapoptotic Bcl-2 proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the German Pharmaceutical Society, DPhG (to V. M. D.). 
2 To whom requests for reprints should be addressed, at Department of Pharmacy, Center of Drug Research, Butenandtstrasse. 5–13, D-81377 Munich, Germany. Phone: 49-89-2180-7161; Fax: 49-89-2180-7173; E-mail: Verena.Dirsch{at}cup.uni-muenchen.de
3 The abbreviations used are: CD95-L, CD95-ligand; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinine; PI, propidium iodide; FACS, fluorescence-activated cell sorter/sorting; afc, 7-amino-4-trifluoromethylcoumarin; DEVD-afc, Asp-Glu-Val-Asp-afc; JC-1, 5,5',6–6'tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide; MMP, mitochondrial membrane permeabilization; m, mitochondrial transmembrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PTP, permeability transition pore; ANT, adenine nucleotide translocator; MAPK, mitogen-activated protein kinase.
Received 2/26/01. Accepted 6/ 1/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. Mol. Pharmacol., 53: 402-407, 1998.This article has been cited by other articles:
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-), cells overexpressing Bcl-xL (Jurkat/bcl-xL, 
) as well as cells overexpressing Bcl-2 (Jurkat/bcl-2, 
). Apoptotic cells were quantified by FACS as described under "Materials and Methods." Data points, the mean ± SE of five (B, top graph) or three (B, bottom graph) independent experiments performed in triplicate.